The reliable and accurate measurement of oxygen supply in mammalian tissue is important to ensure that the oxygen supply is adequate. The circulatory system employs special oxygen-carrying protein molecules in red blood cells (hemoglobin) to deliver oxygen from the lungs throughout the body. Once dissociated from hemoglobin, oxygen is delivered to its consumption sites in cells by diffusion. It is the measurement of this dissolved unbound oxygen that is most critical for quantifying tissue physiological status. The phosphorescence quenching method underpinning this patent application is unique in its ability to accomplish such a measurement. This method is based on use of special phosphorescent probe molecules, which report on oxygen concentration in the environment with high specificity and accuracy.
Measurements of oxygen by phosphorescence quenching provide information on oxygen consumption by the tissue, and through that, allow evaluation of tissue diseased states. Oxygen is a key metabolite, and tissue hypoxia is a critical parameter with respect to various tissue pathologies, such as retinal diseases (Berkowitz et al., Invest. Ophthalmol. Visual Sci. 40:2100-2105 (1999); Linsenmeier et al., Ophthalmol. Visual Sci. 39:1647-1657 (1998)), brain abnormalities (Vannucci et al., J. Exp. Biol. 207:3149-3154 (2004); Brunel et al., J. Neuroradiology 31:123-137 (2004); Johnston et al., Neuroscientist 8:212-220 (2002)), and cancer (Evans et al., J. Appl. Physiol. 98:1503-1510 (2005)). Different oxygen levels in tissues can be indicative of tissue structure abnormalities, defects, whether caused externally, or by genetic manifestations, resulting from disease.
Imaging tissue oxygen in vivo presents a challenging and important problem. Nevertheless, currently developing imaging technologies for mapping tissue oxygenation (Rajendran et al., Radiol. Clin. North Am. 43:169-187 (2005)) (e.g., NMR/EPR (Subramanian et al. NMR Biomed. 17:263-294 (2004)), PET (Piert et al., Nucl. Med. 46:106-113 (2005); Apisarnthanarax et al., Rad. Res. 163:1-25 (2005)), near infrared tomographic techniques (Fenton et al., Brit. J. Cancer 79:464-471 (1999); Liu et al., Appl. Opt. 39:5231-5243 (2000)), etc (Ballinger, Sem. Nucl. Med. 31:321-329 (2001); Foo et al., Mol. Imag. Biol. 6:291-305 (2004)) suffer from many deficiencies, including invasiveness, low spatial and/or temporal resolution, lack of absolute calibration, poor specificity, etc., and remain yet to be adequately developed.
The phosphorescence quenching method (Vanderkooi et al., J. Biol. Chem. 262:5476-5483 (1987); Wilson & Vinogradov, In: Handbook of Biomedical Fluorescence. Mycek M-A, Pogue B W, eds. Marcel Dekker; New York: 2003. Ch. 17) is superior in its ability to directly detect oxygen in tissue. A detailed summary is presented by Vinogradov & Wilson (2012) “Porphyrin-dendrimers as biological oxygen sensors,” In Designing Dendrimers (Capagna, Ceroni, Eds.), Wiley, New York) following the effective filing date of this invention. When a phosphorescent probe is dissolved in the blood and excited using appropriate illumination, its phosphorescence lifetime and intensity become robust indicators of oxygen concentration in the environment. Phosphorescence quenching is exquisitely sensitive and selective to oxygen, possesses excellent temporal resolution and can be implemented for high-resolution hypoxia imaging in 2D (Rumsey et al., Science. 241:1649-1652 (1988); Vinogradov et al., Biophys. J. 70:1609-1617 (1996); Shonat et al., Annal. Biomed. Eng. 31:1084-1096 (2003)).
Efforts to develop 3D near infrared tomographic modality for mapping tissue oxygenation using phosphorescences, include Soloviev et al., Applied Optics 42:113 (2003); Soloviev et al., Applied Optics 43:564 (2004); Apreleva et al., Optics Letters 31:1082 (2006); Apreleva et al., Applied Optics 45:8547 (2006); Apreleva et al., Optics Letters 33:782 (2008), and recent clinically relevant developments of Cerenkov radiation-induced phosphorescence (Zhang et al., Biomedical Optics Express 3:2381 (2012). But highly accurate and versatile methods for measuring oxygen remain to be further developed.
For phosphorescent compounds to be suitable for use as a phosphorescent oxygen probe (aka “phosphor” or “oxyphor”) in determination of tissue oxygenation, it is desirable that the compounds have (1) high absorbance in the near infrared region of the spectrum where natural chromophores of tissue, such as hemoglobin or myoglobin, have only very weak absorption; (2) phosphorescence with high quantum yields at room temperature, preferably greater than 0.05; and (3) suitable lifetimes, preferably from about 0.3 to about 1 msec.
A new class of phosphors suitable for oxygen measurement was previously reported in Vinogradov and Wilson, J. Chem. Soc., Perkin Trans. 2, 103-111 (1995), and in U.S. Pat. No. 4,947,850, “Method and Apparatus for Imaging an Internal Body Portion of a Host Animal,” by Vanderkooi and Wilson), and U.S. Pat. No. 5,837,865, “Phosphorescent Dendritic Macromolecular Compounds for Imaging Tissue Oxygen,” by Vinogradov and Wilson), which are incorporated herein by reference. In the general, the phosphorescent probes for oxygen measurements comprise three functional parts: 1) phosphorescent core; 2) encapsulating and protecting ligands and 3) the hydrophilic outer layer, which is usually made of monomethyloligoethyleneglycol or simply polyethyleneglycol (PEG) residues. Parts 2 and 3 comprise the so-called immediate “surrounding environment” of the phosphorescent core chromophore.
The functions of the three parts are as follows: a) phosphorescent core provides optical signal (phosphorescence), inducible by red/near infrared excitation sources and responsive to changes in the partial pressure of oxygen (pO2); b) encapsulating ligands allow tuning of the core accessibility to oxygen to optimize probe's sensitivity in the physiological pO2 range; and c) outer layer provides solubility and isolates the probe from interactions with endogenous biological species (proteins, nucleic acids, membranes etc) in order to maintain the calibration constants for quantitative pO2 measurements in biological environments.
Both aforementioned patents teach compounds based on complexes of metals, such as Pd and Pt, with porphyrins and aromatically π-extended porphyrins, such as, for example, tetrabenzoporphyrin, tetranaphthaloporphyrin, tetraanthraporphrin and various derivatives thereof, which play the role of phosphorescent cores (part 1). These complexes possess bright room temperature phosphorescence, and Pd and Pt complexes of tetrabenzoporphyrins and tetranaphthaloporphyrins are especially desirable because they show strong light absorption in the near IR region (610-650 nm and 700-720 nm, respectively), where tissue is practically transparent. Moreover, Pd tetrabenzoporphyrins (PdTBP) and their derivatives have been shown to have long-lived phosphorescence (˜250 μsec) with quantum yields of 0.08-0.10%. These values have been later re-measured against improved fluorescence standards used throughout the remainder of this specification, and shown to be 0.0015-0.04 (see Esipova et al, Anal. Chem. 83:8756 (published on-line Oct. 11, 2011).
Generally, the surrounding environment determines properties of the phosphorescent probe with respect to oxygen measurement, including water solubility, toxicity, oxygen quenching constant, sensitivity of the measurements to chemically active components of tissue, and ease of excretion of the probe from the body through the kidney. It is also desirable to design the surrounding environment, such that it comprises an inert globular structure around the phosphor, through which only small uncharged molecules, i.e., oxygen, can diffuse into the close vicinity of the phosphorescent core for efficient quenching.
The '865 patent above teaches that the optimal surrounding environment for the phosphorescent core is made of dendrons as encapsulating ligands (part 2) and polyethyleneglycols (or oligoethyleneglycols) as the outer layer of the probe (part 3). (Note that together the encapsulating dendrons are said to comprise a dendrimer. Accordingly, the corresponding phosphorescent probes are termed dendritic.) Dendritic probes so far have shown to be superior phosphors for oxygen measurements in biological systems. Many laboratories around the world currently use these molecules for oxygen measurements in the blood, tissue interstitial space, various organs and with application of different modes of the phosphorescence quenching method (see above). See, e.g., Sakadzic et al., Nat. Methods 7:755 (2010); Devor et al., J. Neuroscience 31:13676 (2011); Lecoq et al., Nature Medicine 17:893 (2011); and since the effective filing date of this application, Parpaleix et al., Nature Medicine 19:241-246 (2013), which have capitalized on the use of the dendritic oxygen probes to decipher brain energy metabolism in the field of neuroscience. Thus the prior and present art have clearly established tremendous value of dendritic oxygen probes, warranting their further improvement and optimization.
It has therefore been an ongoing need in the art to further improve on the structure of dendritic phosphorescent probes by altering and improving their chemical structure, thereby providing a “next generation” of oxygen sensors with substantially improved phosphorescence emission for better imaging capabilities, ease of use, and range of applicability. In addition, some of the new molecules in this disclosure provide measurements of oxygen, not only in an aqueous environment, but also in liquid organic media, such as organic solvents and/or oils.